This proposal seeks new insight into the fundamental organization of the human cell division cycle and how perturbations to that organization lead to genome instability and pathological states. Complete and efficient duplication of the entire human genome requires that many thousands of DNA replication origins become ?licensed? in G1 of each cell division cycle through the loading of MCM helicase complexes. DNA-loaded MCM complexes are then activated during S phase. Loss of normal origin licensing control causes hypersensitivity to replication stress and induces genome instability, which can ultimately lead to oncogenesis, developmental defects, and degeneration. Our long-term goal is to understand how DNA replication origin licensing control is coordinated with intracellular and extracellular signaling pathways that control proliferation and development. We hypothesize that perturbations to this coordination cause genome instability and proliferation failure. Our experimental approach is a combination of quantitative single cell analyses with molecular biology and biochemistry using cultured human cells. We focus on uncovering molecular mechanisms and dynamics, and then testing the cellular consequences of disrupting those mechanisms. Our recent progress, innovative experimental strategies, and preliminary data inspire a new series of projects to address these specific goals: 1) determine precisely how changes in individual cyclin/CDK enzymes impact origin licensing and the G1 to S phase transition 2) define the molecular consequences phosphorylating an essential origin licensing protein, Cdt1, and 3) define the relationships among CDK-mediated phosphorylation, the APC/C E3 ubiquitin ligase, and origin licensing, particularly for regulation of the Cdc6 licensing protein and preventing re-replication. Success will shed light on the mechanisms that can drive mutagenesis, cancer, cell death, and aging. The deep understanding of cell proliferation control sought through the pursuit of these aims will have downstream implications for efforts to precisely define and treat human disease and for future regenerative therapies.